X4P-001, an Orally Bioavailable CXCR4 Antagonist, Increases Immune Cell Infiltration and Tumor Inflammatory Status in the Microenvironment of Melanoma

Robert H.I. Andtbacka1, Robert H. Pierce2, Jean S. Campbell2, Melinda Yushak3, Mohammed Milhem4, Merrick I. Ross5, Katie Niland6, Lu Gan6, Sudha Parasuraman6, Yan Wang6 1 Surgical Oncology, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT; 2 Fred Hutchison Cancer Research Center, Seattle, WA; 3 Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA;

4 Medical Oncology, University of Iowa, Iowa City, IA; 5 Surgical Oncology, MD Anderson Cancer Center, University of Texas, Houston, TX; 6 X4 Pharmaceuticals, Cambridge, MA

Presented at The Society for Immunotherapy of Cancer Annual Meeting · November 7–11, 2018 · Washington, D.C.

Based on analysis of multiplex IHC staining of tumor samples from patient #5 using HALO software:• X4P-001 increased the percentage of CD4, CD8,

PD-1, and PDL-1 positive cells in the TME• The percentages of Treg (FoxP3 positive) cells

and macrophages (CD68/CD163positive; 24.1% vs. 25.4%; not shown) were not altered

Formalin-fixed paraffin-embedded melanoma samples were stained sequentially with a 6-component immunophenotyping antibody panel, including CD4, CD8, PD-1, PD-L1, macrophage cocktail (CD68 + CD163), and FoxP3. DAPI was used as a nuclear counterstain. Antibodies were detected using HRP-catalyzed deposition of fluorescent tyramide substrates (Opal, Perkin-Elmer). Images were obtained using spectral imaging, autofluorescence subtraction and unmixing (Vectra 3.0, Perkin-Elmer), and analyzed using HALO image analysis software.

Biopsy samples were obtained at baseline (top row) and at the end of X4P-001 monotherapy (bottom row). The left column shows biopsy samples with outlines of normal tissue (yellow) and the tumor border (green). The center column shows the enlarged boxed regions from the left column stained with the markers listed in the figure key. The right column contains higher magnification views of the boxed regions in the center panel. X4P-001 leads to increased numbers of CD3+ cells within tumor borders and decreased expression of VISTA, a check point molecule that inhibits T cell activation and proliferation.

The number of CD8+ T cells at the melanoma tumor interface with normal tissue was quantified using multiplex IHC and HALO image analysis. CD8-labeled cells within 100 µm of the inside or outside of the tumor boundary with normal tissue were counted. The number of CD8+ cells/mm2 was plotted against distance from the boundary in 25 µm bands. After 3 weeks of X4P-001 monotherapy, the total density of CD8+ cells within the boundary area was increased four-fold compared with baseline.

• Treatment with X4P-001 as a single agent and in combination with pembrolizumab is well-tolerated

• X4P-001 monotherapy enhances immune cell infiltration and activation in the TME, as evidenced by:

– Increased IFN-gamma gene expression signature score – Increased Tumor Inflammation Signature (TIS)  – Increased tumor-infiltrating CD8+ T cells and CD8/FoxP3 ratio – Decreased expression of the check point inhibitor VISTA in tumor biopsies 

• Increases in multiple chemoattractant factors in serum were observed after single agent treatment with X4P-001. This observation is consistent with increased trafficking of immune cells post CXCR4 inhibition.

• Mean serum concentration for CXCL9 and CXCL10 are increased after combination treatment by 5.2 and 2.8-fold respectively, consistent with immune stimulation5

• Increased IFN-gamma gene expression signature scores and PD-L1 levels after single agent X4P-001 treatment support the use of X4P-001 in combination with check point inhibitors

Acknowledgements: The authors would like to thank the patients and their families, investigators, co‑investigators, and the study teams at each of the participating centers. Special thanks to Drs. Kam Sprott and Kimberly Smythe for their contributions in IHC assay development and in IHC analysis by HALO™. Special thanks to Dr. Lauri Aicher for her contribution in NanoString Analysis. Disclosure: Study is sponsored by X4 Pharmaceuticals. The Authors received editorial assistance from Acumen Medical Communications. References: 1) Duda DG, Kozin SV, Kirkpatrick ND, et al. CXCL12 (SDF1a)‑CXCR4/CXCR7 Pathway Inhibition: An Emerging Sensitizer for Anticancer Therapies? Clin Cancer Res. 2011;17(8):2074‑2080. 2) Feig C, Jones JO, Kramana, et al. Targeting CXCL12 from FAP‑expressing carcinoma associated fibroblasts synergizes with anti–PD‑L1 immunotherapy in pancreatic cancer. PNAS. 2013;110(50):20212‑20217. 3) Righi E, Kashiwagi S, Yuan J, et al. CXCL12/CXCR4 Blockade Induces Multimodal Antitumor Effects That Prolong Survival in an Immunocompetent Mouse Model of Ovarian Cancer. Cancer Res. 2011; 71(16):5522‑5534. 4) Ayers, Lunceford, Nebozhyn et al. IFN‑γ‑related mRNA profile predicts clinical response to PD‑1 blockade. Clin Invest. 2017;127(8):2930‑2940. 5) Saxena, Wang and Mier. Efficacy and mechanism of action of CXCR4 inhibition in B16 OVA melanoma model. SITC, Nov 2017.

Conclusions

200 µm 200 µm

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CD4 CD8 PD-1 PD-L1 FoxP3 CD4 CD8 PD-1 PD-L1 FoxP3

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Combination Treatment Robustly Increases Serum Concentrations of CXCL9 & CXCL10

X4P-001 Monotherapy Increased the IFN-gamma Signature & Tumor Inflammation Signature (TIS) in the TME

IFN-gamma TIS2.5

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Significant Serum Cytokine and Chemokine Changes at Week 4 of X4P-001 Monotherapy Compared to Baseline

Biomarker Signed Rank Student’s t

Increase

TNF-Related Apoptosis-Inducing Ligand Receptor 3 (TRAIL-R3) < 0.0001 < 0.0001

Interleukin-6 receptor (IL-6r) 0.0002 0.0007

Myeloid Progenitor Inhibitory Factor 1 (MPIF-1) 0.0002 < 0.0001

Tumor necrosis factor receptor 2 (TNFR2) 0.0004 0.0005

Interleukin-2 Simoa (IL-2 Simoa) 0.0006 0.0063

Monokine Induced by IFN-gamma (MIG; CXCL9) 0.0012 0.0194

EN-RAGE 0.0020 0.0041

Tumor Necrosis Factor Receptor I (TNF RI) 0.0021 0.0032

Eotaxin-2 0.0026 0.1533

Chemokine CC-4 (HCC-4) 0.0034 0.0006

Urokinase-type plasminogen activator receptor (uPAR) 0.0034 0.0029

Interleukin-2 receptor alpha (IL-2 receptor alpha) 0.0103 0.0073

Macrophage Inflammatory Protein-1 beta (MIP-1 beta) 0.0103 0.0264IFN-gamma Induced Protein 10 (IP-10; CXCL10) 0.0157 0.10996Ckine 0.0210 0.1296

Macrophage inflammatory protein 3 beta (MIP-3 beta) 0.0353 0.0807

Macrophage-Derived Chemokine (MDC) 0.0353 0.0680

AXL Receptor Tyrosine Kinase (AXL) 0.0463 0.0555

Tissue Inhibitor of Metalloproteinases 1 (TIMP-1) 0.0616 0.0278

Decrease

Plasminogen Activator Inhibitor 1 (PAI-1) 0.0007 0.0007

Brain-Derived Neurotrophic Factor (BDNF) 0.0081 0.0040

Epidermal Growth Factor (EGF) 0.0237 0.0302

E-Selectin 0.0327 0.3136

Monocyte Chemotactic Protein 2 (MCP-2) 0.0377 0.0289

Timepoint ROI Cells/mm2 CD3/mm2 COX-2/mm2 CD206/mm2 VISTA/mm2 B7H3/mm2 CD163/mm2

Day 1Whole Tissue 4149.31 308.53 2.87 259.38 1673.17 2998.14 464.86

Tumor 5758.23 239.91 3.40 264.79 2640.08 4826.36 426.51Non-tumor 2057.63 400.31 2.07 252.20 410.11 615.63 514.86

Week 4Whole Tissue 3572.93 738.82 1.97 182.26 457.53 2288.84 552.27

Tumor 5297.64 1050.68 2.98 161.25 852.86 3952.27 661.21Non-tumor 2217.73 494.97 1.22 199.30 144.72 980.49 467.25

Timepoint CD8 Count CD8 Within Interface Area CD8 Average Distance to Interface (µm) Total Interface Area (mm2) CD8 Average Density (cells/mm2)

Day 1 8924 5233 -21.21 13.7694 380.0449Week 4 25894 7557 -18.36 4.8738 1550.5403

CD163 CD206 VISTA COX-2 CD3 B7H3 DAPI

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Average fold change: 5.16Median fold change: 5.23

Average fold change: 2.83Median fold change: 2.79

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Increased CD8 : FoxP3 Ratio & PD-L1 in TME Post-X4P-001 Treatment

Increased CD3 and Decreased VISTA Post Single Agent Treatment of X4P-001

X4P-001 Monotherapy Increases Intratumor CD8+ Cell Density

NanoString analysis and mIF staining were conducted as described in Methods. Raw counts were normalized using the geometric mean of housekeeping genes. The cytotoxic T lymphocyte (CTL) gene signature was calculated for each patient sample from the geometric mean of normalized counts for CD8A, CD8B, FLTLG, GZMM, and PRF1. The mean was log10-transformed to generate the gene expression score.

Increased Cytotoxic CD8+ T Cells Post-X4P-001 Treatment

CTL Signature Pt #5 Post-X4P-001Pt #5 Pre-dose

CD8CD8

• The CXCR4/CXCL12 pathway plays a central role in the trafficking of key immune cells in the tumor microenvironment (TME).1

• X4P-001 is an oral, selective, allosteric CXCR4 inhibitor. CXCR4 antagonist treatment alone demonstrates robust inhibition of murine B16-OVA melanoma growth.2

• We hypothesize that the disruption of the CXCR4/CXCL12 signaling by X4P-001 will favorably modulate the immune cell profile in the TME and increase CD8+ T cell infiltration, improving responses to checkpoint inhibitors and other backbone therapies.

• A biomarker-driven Phase 1b clinical study was conducted in melanoma patients to test this hypothesis (NCT02823405).

Key Eligibility Criteria

• As of October 3, 2018 16 patients have been enrolled, and biopsies from 13 patients have been analyzed. Nine had both pre-dose and post-X4P-001 treatment-evaluable biopsies. The biopsy analyses focused primarily on post-X4P-001 monotherapy comparisons to baseline due to limited sample availability post-combination treatment.

• Multiplex immunohistochemistry (IHC) images were acquired on a Perkin Elmer Vectra 3.0 (6-plex images) or an Aperio FL (3-plex images). All images were analyzed using Indica Labs HALO™ analysis software (High-plex and Cytonuclear FL modules).

• Sera was prepared from collected blood samples, and the concentrations of panels of chemokines, cytokines, and growth factors were measured using the Multi-Analyte Profile platform (Myriad RBM). The proteins that were examined include the ones in HCANCER2, HMP8, HMPC19, HMPC42 MAPs and IL15, IFN-gamma, and IL2 by Simoa.

• RNA was extracted from formalin-fixed paraffin-embedded (FFPE) slides and analyzed using the PanCancer Immune Profiling and PanCancer Progression Panels supplemented with 30 user-defined genes (Nanostring Technologies). Raw counts were normalized using the geometric mean of housekeeping genes, and the normalized data from both panels were merged and analyzed with nSolver software (Version 4.0). The tumor inflammatory signature (TIS) was calculated from 18 genes by taking the log10 of the geometric mean of the normalized counts across each gene set to generate a “gene signature score”.3 The interferon-gamma (IFN-gamma) gene signature was determined from the normalized counts of six genes (IFN‑gamma, CXCL9, CXCL10, HLA‑DRA, IDO1, and STAT1), as described by Ayers et al.4

• X4P-001 was well-tolerated as monotherapy and in combination with pembrolizumab• Adverse Events (AEs) assessed as related to X4P-001 during monotherapy (> 15%) was diarrhea (31%)• AEs assessed as related to either X4P-001 or pembrolizumab (> 15%) at any time were

diarrhea (44%), fatigue (38%), rash macro-papular (25%), and dry eye (19%)• No X4P-001 related Grade 3 AEs were observed during the monotherapy period• Grade 3 AEs assessed as related to either X4P-001 or pembrolizumab at any time were rash

maculo-papular (13%), hypertension, acute kidney injury, ALT increased, AST increased, blood bilirubin increased, diarrhea, immune-mediated adverse reaction, and stomatitis (6% each)

• There were no Grade 4 or Grade 5 AEs at any time during the study

Background

Safety

Study Design

Methods

Demographics and Baseline Characteristics

Pembrolizumab (pembro) treatment2 infusions (2 mg/kg)

X4P-001 (400 mg QD)

Tumor Biopsies &Blood Samples

* The first patient was dosed at 200 mg BID

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Post-X4P-001(Monotherapy)

Post-Combination(X4P-001 + pembro)

End of Treatment(EOT)

3 weeks 3 weeks 3 weeks

*

• Mean patient age was 74.6 (± 9.6 years); the median age was 74.5 (range 53-91 years)• Of the 16 patients enrolled, 10 (62.5%) were male and 6 (37.5%) were female• 15 patients (94%) were White and 1 (6%) was Asian• 9 patients (56%) had a screening ECOG status score of 0 and 7 (44%) had a score of 1• At study entry, 4 patients (25%) were stage IIIB, 10 patients (62.5%) were stage IIIC, and 2 patients

(12.5%) were stage IV M1A

Inclusion:• ≥ 18 years of age • Histologically confirmed malignant melanoma• ≥ 2 separate cutaneous or subcutaneous lesions suitable for biopsies (≥ 3 mm)Exclusion:• ECOG Performance Status ≥ 2 • Prior checkpoint inhibitor therapies (anti-CTLA-4, PD-1, PD-L1) or oncolytic virus therapy• Ongoing HIV, hepatitis C virus, or uncontrolled infection• Occurrences of myocardial infarction, ≥ Grade 3 hemorrhage, chronic liver disease, or other active

malignancies in the past 6 months

Day 1

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Recruitment ofTregs and MDSC

Decreased Traffickingof Tregs and MDSC;

Increased CD8+ T cell Influx

Immune suppressionBlood supplyTumor growth

Immune suppressionBlood supplyTumor growth

CXCR4CXCL12 X4P-001

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Tumor CellCancer-AssociatedFibroblast (CAF) Treg MDSC CD8+ T cells

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